Color vision is an important aspect of our perception of the world, enhancing our recognition of objects in complex visual scenes and allowing us to assign them an identity and quality. How are colors encoded in the brain? Despite decades of research, this question remains unanswered. It is widely accepted that color opponent neurons, responding with opposite polarity to wavelengths in different parts of the spectrum, are the building blocks for color vision. However, how color opponent neuron signals are combined to give rise to hue-specific neurons, with narrow spectral sensitivity, and how these neurons contribute to color perception is unknown. Analyses of color circuits in a genetically tractable organism are critical to answering these questions. Drosophila melanogaster provides a powerful system to investigate how a compact brain solves the problem of color coding, combining genetic access to cell-type-specific neural populations, a well-defined neural anatomy, and sophisticated behaviors. Moreover, vertebrate and invertebrate visual systems present many functional similarities. The fact that these diverse systems show convergence in solutions to visual processing problems motivates our investigation in a simple model, with the intention of extracting fundamental principles of relevance to mammalian systems. Fruit flies are capable color discrimination and have the hardware necessary for wavelength comparison: four types of cone-like photoreceptors each expressing a unique narrow-band rhodopsin of different wavelength sensitivity, ranging from UV to green. However, the way spectral information from these photoreceptors is processed in the brain is unknown and is the focus of this proposal. We will use genetic neural manipulation techniques, in vivo two-photon imaging, electrophysiology, and behavioral assays augmented by quantitative analysis and modeling, to identify the computational algorithms and neural mechanisms that govern color vision. Aim 1 will ask what kind of spectrally opponent mechanisms exist in Drosophila and determine the identity of neurons and synaptic interactions in the underlying circuits. We will, in addition, generate tools to disrupt color opponent signals. Aim 2 will use connectomics data in conjunction with tracing methods to define and functionally characterize color circuits postsynaptic to color photoreceptors. We will investigate how color-opponent signals are integrated to give rise to higher order color neurons. Aim 3 will characterize how the response of neurons in these circuits support both innate and learned color-guided behaviors, marking an experimental effort to draw a causal link between color opponency, color circuits and color perception, an approach that has been difficult in classical, non-genetically tractable, models for color vision. These studies will provide a detailed understanding of how spectral information is processed in the fly brain and serve as a guide to investigate wavelength computations underlying color vision in the brain of more complex animals.